Cloning, expression, purification and characterization of the alternate splice Src variants for drug discovery

Protein tyrosine kinases (PTKs) are key members of intra- and extra-cellula r signaling pathways. Aberrant signaling pathways are responsible for many human diseases, making these enzymes targets for drug development programs. The difficulty in PCR-amplification of Src due to the high G-C content was overcome using a commercial "G-C melt" reagent. The N06 Src was cloned alo ng with the N12 and N23 neuronal variants. Neuronal variants of Src occur d ue to splicing within the N-loop of the SH3 domain. These variants have gre ater catalytic activity. Affinity purification methodologies were establish ed that takes advantage of binding sites within the Sill and SH2 domains. T he purified enzyme is stable, without loss of activity for >1 year when fro zen and more than 1 week at 4 degreesC. A 96-well solution phase assay was developed and validated that overcomes many of the false positives and nega tives generated by other assays. Studies of the catalytic mechanism have in dicated that a second metal ion is essential for catalysis. Some transition metals can be substituted for the second metal ion and maintain activity w hile others act as dead-end inhibitors with binding constants in the sub-mi cromolar range. The precise role of this second metal ion is being studied. (C) 2001 Elsevier Science B.V. All rights reserved.